1. Field of the Invention
This invention relates to a method for quantifying total mRNA in a biological sample such as crude cell lysates containing cytosolic RNA, and particularly to that for quantifying total mRNA by using a microtiter plate to which poly(A)-complementary oligonucleotides are immobilized, which method allows for a rapid, accurate, and nonradioactive quantification of total mRNA even when rRNA (ribosomal RNA) and tRNA (transfer RNA) are present in the sample. This invention also relates to a rapid chemosensitivity determination method using the total-mRNA measuring method.
2. Background of the Art
New gene sequences are discovered daily, and advanced molecular biological techniques are revolutionizing clinical practice in genetic disorders, oncology, infectious diseases, etc. Although the current major focus is set on use of DNA to identify disease genes, genetic mutations, translocations, or foreign genes as infectious agents, the analysis of specific mRNA also attracts clinical scientists who may wish to quantify specific gene expression in certain tissues and cells during the course of disease, both before and after various treatments.
Technologies are available for the analysis of mRNA, e.g., Northern blotting (Sambrooke et al., Molecular cloning, a laboratory manual, 2nd ed., Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press, 1989:7.28-7.52) and reverse transcription followed by polymerase chain reaction (RT-PCR: reverse transcription-polymerase chain reaction) (Kawasaki et al., Erlich HA, ed. PCR technology, New York, Stockton, 1989:89-97), and in each assay, positive signals can be quantified by various techniques. However, the comparison of positive signals among different clinical specimens is still quite difficult, given the lack of normalization procedures. For example, if specific mRNA expression in cancers is compared among different patients, such signals can be expressed in terms of wet weight, protein concentration, DNA content, RNA content, etc. The most common practice in Northern blotting is to apply the same amounts of RNA and confirm that ribosomal RNA (rRNA) signals of the same intensity are observed on the same filters (Okamoto et al., Biochem Biophys Res Commun 1993; 197:878-85). However, because the mRNA content accounts for less than 5% of total RNA or rRNA, the same amount of measured total RNA or rRNA does not indicate that the amount of applied mRNA is equal among tested samples.
Alternatively, one can compare specific signals with the expression of other, known housekeeping genes, e.g., .beta.-actin (Ponte et al., Mol Cell Biol 1983; 3:1783-91) and glyceraldehyde-3-phosphate dehydrogenase (Tso et al., Nucleic Acids Res 1985; 13:2485-502). However, the expression of these genes is also known to vary substantially under certain conditions. The most practical solution is to purify mRNA and use the same amount of mRNA for Northern blots or for RT-PCR, even though purification of the mRNA requires additional time-consuming steps.
Other attempts have been made to quantify the amount of total mRNA in test samples. In a classical approach, poly(A)+ mRNA is purified from test samples and the final amount of purified mRNA is determined by measuring A.sub.260 ; however, this method requires a relatively large amount of starting material and multiple time-consuming steps. Johnson, et al. (Johnson et al., Cell 1974; 1:95-100 and Johnson et al., J Cell Biol 1976; 71:933-8), in a series of studies, chase-labeled mRNA with radioactive mononucleotides and determined the radioactivity of the purified RNA or mRNA. However, this method requires radioactive materials and chase-labeling mRNA, thereby providing non-absolute amount of mRNA. In flow cytometry, both DNA and RNA contents from acridine orange-stained cells were simultaneously determined by two-color analysis: green emission for DNA and red emission for RNA (Traganos et al., Cytometry 1982; 2:212-8 and Hadjlssotiriou et al., Br J Urol 1985; 57:668-75). However, mRNA cannot be determined by this method, since the dyes stain not only mRNA but also rRNA and tRNA. Although Harley (Harley CB, Gene Anal Tech 1987; 4:17-22) reported a quantitative method of measuring total amounts of mRNA by hybridizing radiolabeled oligo-(dT) or RNA on nitrocellulose membranes, this method required radioactive probes and a lengthy process such as probe hybridization, washing, and detection). Further, the assay is semiquantitative; it is uncertain whether the applied RNA samples are entirely immobilized on nitrocellulose membranes and available for hybridization to each probe.
Previously, we introduced a unique research system involving microtiter plates to which oligonucleotides containing oligo-(dT) sequences had been immobilized covalently (GenePlate.TM.: Mitsuhashi et al., Nature 1992; 357:519-20). Furthermore, we also reported that DNA and RNA in solution (Ogura et al., BioTechniques 1994; 18:231-2), and oligonucleotides immobilized on a microtiter plate (Ogura et al., BioTechniques 1994; 18:1032-4) could be quantified by adding the fluorescent indicator dye YOYO-1.TM. (Glazer et al., Nature 1992; 359:859-61). However, heretofore, oligonucleotides have never been quantified with an indicator such as YOYO-1.TM. while being hybridized with oligonucleotides immobilized on a microtiter plate, because specificity and sensitivity of indicators were believed uncertain. In addition, it was difficult to separate signals of mRNA from those of rRNA and tRNA, leading to overestimation of total mRNA.
Biological significance of total mRNA has been reported: The amount of total mRNA in rapidly growing cells was significantly higher than that of resting cells (Johnson et al., Cell 1974; 1:95-100). However, measuring total mRNA has not been applied to medical or diagnostic use such as a chemosensitivity test in a practical manner, because rapid and sensitive measuring methods are not available.
To select the most appropriate anticancer drugs and their optimum doses, various chemosensitivity tests have recently become available, which include the method of identifying dead or dying cells by measuring increased cellular permeability (Ross et al., Cancer Res 1989; 49:3776-3782), the measurement of DNA synthesis using .sup.3 H-thymidine incorporation (Kern et al., Cancer Res 1985; 45:5346-5441), the measurement of cellular metabolic activities using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Mosmann et al., J Immuno Methods 1983; 65:55-63), AlomarBlue (Fields et al., American Biotechnology Laboratory, March 1993 and de Fries et al., J. Clin Lab Anal 1995; 9:89-95), etc. However, these assays require cell culture for at least a few days to detect the cytotoxic effect of anticancer drugs, although some cancer cells are extremely difficult to maintain in culture. Furthermore, because cellular phenotype may change significantly during culture conditions, the results of long culture may not correspond to the results in vivo (Bellamy et al., Drugs 1992; 44:690-708).